While antiestrogen therapies improved prognoses of estrogen receptor positive (ER+) breast cancer patients, disease recurrence remains a major problem. Over 50% of ER+ breast cancer cases are intrinsically resistant or develop resistance to become incurable. Common mechanisms of antiestrogen resistance are: deregulation of ER signaling; downregulation of ER itself; and altered drug metabolism. Studies to overcome antiestrogen resistance uncovered contributing roles of growth factors and protein kinases, but targeting these pathways yielded only marginal improvements in clinics. Recently, a CDK inhibitor Ibrance was approved to treat advanced ER+ breast cancer, however it is useful for patients who never received antiestrogen therapy. Hence, nothing holds promise for recurred breast cancer patients. Recently, we reported that G1P3, an interferon and estrogen induced survival protein, plays a critical role in tamoxifen (TAM) resistance in ER+ breast cancer cells. We and others also uncovered the association between elevated expression of interferon-stimulated genes and poor prognoses in breast cancers. These results suggest that genes not yet associated with antiestrogen resistance may drive it. To improve suboptimal therapeutic outcomes in recurrent breast cancers, this proposal aims to identify and validate genes driving antiestrogen resistance. Since breast cancer exhibits heterogeneous molecular and clinical phenotypes, identifying and validating therapeutic targets using conventional methods may take considerable effort and time. To overcome this, our program uses a novel high-throughput, genome- wide, cell-based functional genomics screen called GRIP (genome-wide random insertion of promoter) for rapid identification of targets to overcome antiestrogen resistance. GRIP has potential to activate virtually any gene within the genome of breast cancer cells. The fundamental hypothesis of this proposal is that elevated expression of the gain-of-function genes that complement estrogen-signaling pathways confers antiestrogen resistance in ER+ breast cancer cells. We will test this hypothesis by completing two specific aims. By screening four GRIP libraries of one million ER+ breast cells each for TAM resistance, Aim 1 will discover antiestrogen resistance drivers in functional genomics screens. We postulate that only GRIP activated genes that complement estrogen signaling will result in TAM resistant MCF-7 cells, an estrogen dependent ER+ breast cancer cell line. By assessing the correlation between expression of the identified genes and clinical outcomes in a retrospective clinical study, Aim 2 will define the clinical relevance of GRIP activated genes driving antiestrogen resistance. Major innovations of this project are (1) use of an unbiased, cell-based, functional genomics screen to identify genes that drive antiestrogen-resistance; and (2) rapid establishment of the clinical relevance of antiestrogen resistance genes as therapeutic targets and/or prognosticators. Discovering genes involved in antiestrogen resistance will elucidate the pathophysiology of ER+ breast cancer recurrence and help design strategies to eradicate resistant tumor cells for a lasting curative effect.